In recent years, a large volume of literature has evolved concerning the use of small peptide substrates for the assay of specific proteinases. These substrates usually contain a chromogenic or fluorogenic group which is released upon hydrolysis by the proteinase whereupon the rate of appearance of color or fluorescence can be used to quantify the amount of proteinase enzyme present. This development has made it possible to assay individually many of the serine proteinases involved in the blood coagulation cascade. Peptide substrates specific for thrombin and factor Xa have been used to develop indirect solution assays for the clinically important blood coagulation inhibitor antithrombin-III (AT-III), which is thought to be the major inhibitor of thrombin and factor Xa in plasma. These assays rely on the fact that AT-III is a rapid inhibitor of thrombin in the presence of heparin. The inhibition takes from 10 to 60 minutes in the absence of heparin but only 15 to 60 seconds in its presence. The reaction sequence in these assays is represented by the following equations: ##STR1## The residual thrombin is then assayed using a suitable synthetic peptide substrate. For the assay of AT-III, heparin is provided in excess of the catalytic amount required so that all of the AT-III present in the assay can rapidly exert its thrombin inhibiting effect. To determine the amount of heparin necessary, a titration experiment with increasing amounts of heparin could be run to determine the amount necessary to give the desired result. In the experiments described herein, this assay was not performed, but instead the amounts of heparin used in similar samples diluted for a solution assay were used. It is reported by Blombock, et al [Throm. Diath. Haemouh. 9, 368 (1963)] that the inhibition of thrombin was constant between 2 and 16 U/ml of heparin. In all of the published assays for AT-III there is a time delay after reaction (2) which preceeds the addition of a synthetic peptide substrate to assay the residual thrombin. It has been discovered that this delay is necessary because the heparin catalyzed reaction of AT-III and thrombin proceeds very slowly in the presence of substrates which have low Km values. In many of the assays, an additional constituent, polybrene, is added along with the peptide substrate to neutralize the heparin and prevent any further fast inhibition of thrombin during the assay of residual thrombin. The use of polybrene or another heparin neutralizing substance was initially tried with assays that employ chromogenic thrombin substrates having rather high Km values. This was necessary because the substrate does not compete well with any unreacted AT-III.heparin complex and further inhibition could occur during the hydrolysis step. This practice was carried over into some assays in which chromogenic substrates having lower Km values were employed. The substrate used in the work reported in the following examples, S-2238, has a very low Km value and it was found that polybrene was not necessary.
An assay of the type above is described by Odegard, et al in Thrombosis Research, Vol. 6, pages 287-294 (1975) Pergamon Press, Inc. This assay involves mixing 100 microliters (.mu.l) of test plasma with 2.9 milliliters (ml) of buffer containing heparin whereupon 400 .mu.l of this dilution is trasferred into each of 2 glass tubes and prewarmed in a water bath at 37.degree. C. for 2 to 6 minutes. At this point, 100 .mu.l of a thrombin solution containing 30 NIHU/ml is blown into the tube, and after exactly 30 seconds, 300 .mu.l of a substrate-polybrene solution is blown into the reaction mixture. The chromogenic substrate used is the tripeptide Bz-Phe-Val-Arg-pNA.HCl. Exactly 60 seconds after addition of the substrate, the amidolysis is quenched by blowing 300 .mu.l of acetic acid into the mixture. The optical density is read at 405 nanometers (nm) against a reagent blank containing 400 .mu.l standard plasma dilution, 300 .mu.l acetic acid, 300 .mu.l substrate-polybrene solution and 100 .mu.l thrombin solution mixed in this order. Two optical density readings are taken and their mean is used for reading the AT-III concentation (heparin cofactor activity) from a standard curve.
While this wet assay method is more convenient than earlier assays which involve clotting of fibrin, it is still somewhat time consuming and can be susceptible to human error. A test strip for the quantitative determination of AT-III based on the interaction of thrombin and a chromogenic or fluorogenic substrate would be desirable. However, any increase in speed or convenience obtained by employing a strip containing thrombin and the substrate in close proximity to each other would be more than offset by the fact that the presence of the substrate inhibits the reaction between thrombin and AT-III even in the presence of excess heparin.